Psoralens are linear furocoumarins with the ability to crosslink DNA strands upon photoactivation. A number of different analytical techniques have been devised using this property to form covalent bonds between single-stranded nucleic acid probes containing a base sequence complementary to the base sequence of a nucleic acid target molecule.
A number of synthetic techniques have been devised to prepare nucleosides monoadducted to psoralens for use in these assays. For example, thymidines monoadducted to psoralen, 8-methoxypsoraen, 4,5',8-trimethylpsoralen, and 4'-hydroxymethyl-4,5',8-trimethylpsoralen have been prepared by reaction of these psoralen derivatives with deoxyribonucleic acid followed by enzymatic or chemical hydrolysis of the DNA and chromatographic isolation of the thymidine:psoralen monoadduct. Alternatively, the thymidine:8-methoxypsoralen monoadduct has been prepared from the monomers by irradiating a thin film of the two compounds mixed together. The uridine:4'-hydroxymethyl-4,5',8-trimethylpsoralen monoadduct has been prepared by reacting 4'-hydroxymethyl-4,5',8-trimethylpsoralen with ribonucleic acid followed by the enzymatic or chemical hydrolysis of the RNA and chromatographic isolation of the uridine:4'-hydroxymethyl-4,5',5-trimethylpsoralen monoadduct.
A problem that exists with current photochemical synthesis of adducts is that a mixture of products is produced by current techniques. For example, the photoreaction of 8-methoxypsoralen (1) with 2-deoxythymidine (2) gives a mixture of mono- and di-adducts. ##STR1## The major monoadduct (with cis-anti stereochemistry) and two minor monoadducts are derived by (2+2) cycloaddition involving the 3,4-double bond of 8-methoxypsoralen (pyrone-side). Two monoadducts, one with cis-syn stereochemistry and the other with trans-stereochemistry, are formed by (2+2) cycloaddition of the 4',5'-double bond (furan-side). The minor cis-syn, furan-side product (3) is the most desirable product for use in preparing photocrosslinkable nucleic acid probes. ##STR2##
Although separation of the mixture has been achieved, separation is difficult. Accordingly, there remains a need for a synthetic technique which will provide the desired isomer in increased yield and with a simplified course of purification.